This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific Aims Our Aims for this phase of the project were to: 1) complete the upshift-downshift experiment to define the critical window when perchlorate disrupts sexual development of stickleback fish, 2) complete the dose-response experiment to determine the lowest adverse effect level and the highest no adverse effect level for disruption of gonadal development by perchlorate, 3) validate endocrine assays with the stickleback model, and 4) test whether perchlorate induces spiggin (the male glue protein) production in females, which would indicate an androgen-mediated mechanism of developmental disruption. B. Studies and Results Aim 1 (upshift-downshift experiment): Growing stickleback in the various perchlorate concentrations over the various time windows of exposure was completed in June, 2009 (when the fish were 11 months old). At that point, a subsample of fish from each treatment condition was fixed for histology, with matched tissues prepared for genetic analysis of genotypic sex. The remaining fish have been maintained alive in their treatment condition so that further samples are available from each treatment should sample sizes need to be increased or other tissues need to be examined. The preparation of histological slides is on-going, and should be complete by January, 2010. Preliminary results of thyroid tissue showed marked abnormalities, but gonadal tissue has not yet been examined. Aim 2 (dose-response experiment): Growing stickleback in the various perchlorate concentrations for 11 months in the dose-response experiment was completed in June, 2009. At that point, representative samples of fish from all treatments were fixed for histology, while the remainder were preserved for endocrine analyses. The preparation of histological slides is on-going, and should be complete by January, 2010. Aim 3 (validation of endocrine assays): We have developed and optimized assays of vitellogenin, 11-ketotestosterone, 17b-estradiol, and spiggin, and the validation process is underway for thyroxine and cortisol. The endocrinology lab was moved into the new Conoco-Phillips Integrated Science Building (CPISB), and the functioning of all moved equipment has been validated and calibrated, when applicable. Aim 4 (test for spiggin production in females exposed to perchlorate): We have moved into "production mode" for the spiggin analysis, which has been completed for all of the surplus dose-response fish, making ours the only laboratory in the United States currently performing spiggin analysis. Females exposed to perchlorate do not have spiggin induction, so it appears that their masculinization due to perchlorate is not mediated directly by androgens. C. Significance Achieving the aims outlined above allows us to pursue the Aims of our newly funded RO1 proposal by providing the background data essential for our RO1 experiments. The RO1 experiments will address the question of whether the effects of perchlorate on sexual differentiation are mediated through the thyroid, or whether they are generated by a mechanism that is thyroid-independent (RO1 Aim 1). The experiments will also test specifically the roles of the presumed sole target for perchlorate, NIS, as well as its paralogs, in the gonadal phenotype provoked by perchlorate (RO1 Aim 2). Finally, experiments will take an unbiased genome-wide viewpoint in an innovative use of emerging technology to identify new genes and gene pathways involved in sex differentiation that are disrupted by perchlorate exposure (RO1 Aim 3). Because of the strong conservation of endocrine, genetic, and developmental mechanisms among all bony vertebrates, our results will advance our understanding of human pathologies and disease states that may be exacerbated in sensitive genotypes by perchlorate, a toxicant ubiquitously found in our water and food. Because perchlorate exposure may contribute to the growing epidemic of reproductive disease which is driving down sperm counts and increasing rates of testicular dysgenesis in people living in the United States and other industrial countries, the results of the proposed experiments will be strongly relevant to human reproductive health. D. Plans We expect to complete histological slide preparation in January, 2010 for both the upshift-downshift study and the dose-response study. The upshift-downshift results will provide the data needed for the timing of exposure, while the dose-response study will provide the minimal doses needed for disruption of gonadal development. We plan to begin setting up the new CPISB Vivarium space over the next 2 months to accommodate our fish husbandry needs. In addition, we plan to continue and finalize the laboratory endocrinology by optimizing and validating assays for cortisol and thyroxine as has been completed for the other hormones and proteins. And finally, we will begin to link assay results from the spiggin analyses to concentrations and durations of perchlorate exposure. By the end of this round of INBRE support, we feel confident that we will be fully prepared to begin the RO1 experiments as the NIH funding becomes available.